ES2259674T3 - LIPOSOMAS OF ENCAPSULATION OF ANTI-CANCER DRUGS AND USE OF THE SAME IN THE TREATMENT OF MALIGNOUS TUMORS. - Google Patents

Abstract

Liposomes encapsulating anticancer drugs and their use in the treatment of malignant tumors. The liposomes are covered by a lipopeptide consisting of three substructures: a lipid fragment, an active oligopeptide and a spacer oligopeptide between the other two fragments. Applicable in intravenous administration for the treatment of malignant tumors.

Description

Drug encapsulation liposomes anticancer drugs and their use in the treatment of malignant tumors.

The present invention relates to providing a anti-cancer treatment system that can destroy selectively cancer cells within a living being without affect the remaining cells of the treated organism. Plus specifically, the invention relates to liposomes containing anticancer drugs useful in the treatment above mentioned.

State of the art

Cancer is one of the most diseases extended in developed countries and, specifically, the Tumor metastases are the leading cause of mortality in patients with solid malignant tumors. They consist of the appearance of a new cancerous center, which starts from a primary tumor, in another different organ or tissue. This metastatic process includes a series of sequential stages in which tumor cells they have to interact with cellular components and tissues from the host. These stages are as follows: separation of tumor cells of a primary tumor; invasion of space intravascular; migration through the lymphatic or vascular system towards other tissues; adhesion to the vascular endothelium; extravasation e invasion of the new tissue; and secondary tumor formation.

Throughout this process, the cells Metastatic tumors interact with the components of the extracellular matrices and, specifically, with membranes basals, through their adherence to them, causing their deterioration to through the action of proteolytic enzymes produced by them themselves and / or by the same host cells, stimulated by the tumor cells. Therefore, the alteration of the properties of Cell adhesion is an indispensable element for the appearance of metastasis, since it is a process linked to cell release from the initial tumor, to its migration and to its implantation in new tissue.

The main adhesion molecules involved in this interaction are integrins. Integrins are a family of transmembrane glycoproteins, formed by two α and β chains linked by non-covalent (hydrophobic) bonds. Among other functions, integrins act as receptors for certain extracellular matrix proteins, such as laminin, fibronectin, vitronectin and collagen (Ruoslahti, E., Giancotti, FG, Cancer Cells (1989), 1, 4, 119 to 126). Recently, a change in the expression of integrins in tumor cells has been demonstrated, so their presence in this type of cells has increased (Dedhar, S., Saulmier, R., Cell Biol . (1990), 11, 481 to 489). This increase is the factor responsible for adhesion to the extracellular matrix and the acquisition of metastatic potential.

The main treatment used for the removal of tumors is the administration of cytostatic agents intravenously, particularly those belonging to the anthracycline family (Young, RC, Ozols, RF, Myers, CE, N. Eng. J. Med . (1981 ), 305, 139 to 153). However, due to their lack of selectivity with respect to tumor cells, the development of resistance to these drugs by malignant cells and the different response of the primary tumor and metastasis with respect to their action, this type of treatment normally originates the occurrence of serious side effects, some of which are irreversible or chronic in nature.

Therefore, the main objective of Current chemotherapy focuses on achieving antitumor efficacy improved and reduce the toxicity of these drugs. a way to doing this could consist of taking the cytostatic agent, in a adequate concentration, to reach the target cells, giving as result the selective destruction of the primary tumor or the metastasis without any healthy cell being affected. In this way there would be an increase in the therapeutic index of this drug and They would achieve more effective treatments.

In these systems, the drug is incorporated into the aqueous spaces of the liposome when it is hydrophilic or distribute between these and the lipid bilayers if it has a character more lipophilic Once the drug is encapsulated, it can be Administer to the patient under treatment.

Several researchers have shown that the use of liposomes for antineoplastic administration often improves traditional methods of administration, see, for example: Gabizon et al .: Cancer Res. (1982) 42, 4734-4739 and Van Hossel et al . : Cancer Res. (1984) 44, 3698 to 3705.

It has been observed, through the use of several animal models, that the encapsulation of doxorubicin in liposomes significantly reduces the side effects of toxicity; both chronic and acute. See, by way of example, Rahman et al .: Cancer Res. (1980) 40, 1532-1537, Forssen et al .: Proc. Natl Acad. Sci. USE (1981) 78, 1873 to 1877, Olson et al .: Eur. J. Cancer Clin. Oncol. (1982) 18, 167 to 176, Rahman et al .: Cancer Res. (1985) 45, 796 to 803 and Gabizon et al .: J. Natl. Cancer Inst. (1986) 77, 459 to 467. Additionally, other toxicity indicators, such as alopecia, weight loss, nausea, vomiting, and also dermal necrosis due to extravasation with the administration of doxorubicin in liposomes, can be significantly reduced. Forssen et al .: Cancer Treat. Rep. (1983) 67, 481-484; see also the bibliography cited earlier in this paragraph.

It has also been established in several tumor models that this significant reduction in toxicity does not occur at the expense of a decrease in antitumor efficacy. In addition to the literature cited above, see Rahman et al .: Chemother. Pharmacol (1986) 16, 22 to 27, Gabizon et al .: Cancer Res. (1983) 43, 4730 to 4735 and Br. J. cancer (1985) 51, 681 to 689, Mayhew et al .: J. Natl. Cancer Inst. (1987) 78, 707 to 713, Forssen et al .: Cancer Res. (1983) 43, 546 to 550, and Storm et al .: Cancer Res. (1987) 47, 3366 to 3372.

Given the incidence and characteristics special cancerous metastases, treatment antimetastatic is one of the fields in which more has been invested effort in the search for new alternatives to treatments conventional. Due to its mechanisms of action, its high efficiency proven and its high toxicity, anthracyclines constitute the most studied family of cytostatic agents in the field of encapsulation of drugs in sustained release systems such such as liposomes, which can be seen by the number each time major patents that have been appearing under this heading, of which 80% of the total existing for liposomes are apply to cytostatic agents.

The first generation of liposomes containing anthracyclines corresponded to vesicles formed by PC, PG and cholesterol, in the aqueous interior space of which the drug was encapsulated. These liposomes showed a decrease in the toxicity of the drug, although their antitumor activity was not greater than that of the free drug, they only improved the activity of the drug in the case of tumor models in which metastatic cells propagated through the liver, the most accessible organ for liposomes, but not when tumor growth is local (Mayhew, E., Rustum, Y., Biol. Cell. (1983), 47, 81 to 86). In addition, due to its rapid capture by macrophages of the endoplasmic reticulum, its permanence in the organism was reduced after intravenous injection to a few hours. For this reason, and despite the intensive research carried out, there has been no formulation to meet the expectations initially placed in liposomes as transporters of cytostatic agents. It was during the 1980s when the situation changed with the appearance of the first publications that described that liposomes had glycolipids (Allen, TM, Hansen, C., Rutledge, J., Biochem. Biophys. Acta (1989), 981, 27 to 35; Mori, A., Klivanov, AL, Torchilin, VP, Huang, L., FEBS Lett. (1991), 284, 263 to 266) or hydrophilic polymers such as polyethylene glycol (PEG) (Blume, G. , Ceve, C., Biochem. Biophys. Acta (1990), 1029, 91 to 97, Allen, TM, Hansen, C., Martin, F., Redemann, C., Yau-Young, A., Biochem. Biophys Acta (1991), 1066, 29 to 36) on its surface in order to increase its circulation time in the bloodstream, thus obtaining the so-called "second generation liposomes" or "phantom liposomes"("stealthliposomes") . It seems that this stabilizing effect of PEG and glycolipids is due to their hydrophilic properties that prevent them from forming aggregates on the surface of the liposome and allows it not to be recognized as a ligand of any cellular receptor or of any plasma protein. In addition, its presence on the surface of the liposomes produces a steric effect, since it hinders the action of opsonins and other blood proteins, and reduces the access of macrophage receptors to phospholipid phosphate groups, which gives as result in an increase in blood circulation time.

Subsequently, some authors found that it was possible to improve the characteristics of stability and efficiency of these liposomes by incorporating additives that inhibited lipid peroxidation such as vitamin E acetate, BHT or chromane derivatives (documents EP-0274174, WO-8500968, WO-9202208 and US-5605703).

Despite the fact that these galenic forms they offered a number of benefits over conventional forms, the possibility of directing the vesicles to the target cells in order to improve efficacy with doses of Smaller drug and suppress or reduce the effects secondary.

The basic idea is to incorporate on the surface of the liposome any chemical body that can be recognized selectively by the target cells. Success in leading liposomes towards the target cells reside in an appropriate choice of The vector molecule.

The process of selecting any structure chemistry that can direct liposomes to tumor cells It is not trivial, since specifically in the case of cells tumor, there is a great diversity of proteins located in the membrane, as well as surface antigens and receptors that vary according to metastatic potential, proliferative activity and the tissue in question, in a way that, although they could be used as a basis for selecting recognition structures, in the Practical choice is not immediate. In addition, in many cases, the Authentic situation is that tumor cells and / or proliferative they have as a differentiation characteristic the overexpression of certain structures with respect to normal cells.

The accumulated knowledge to date indicates that adhesion processes and participating proteins they play an essential role in the development of processes metastatic and vascularization necessary for proliferation mobile.

Among the proteins that participate most in these mechanisms, laminin has proven to be a good candidate to direct liposomes, as it has been demonstrated so conclusively that its receptors are overexpressed in cells Tumor

Laminin is the major component of the basement membrane of cells after collagen. Is a glycoprotein formed by three polypeptide chains: α (440 kDa), β (200 kDa) and γ (220 kDa), which are arranged in the Shape of a cross, with two short arms and a long arm. The links between the chains are formed by disulfide bonds and through non-covalent interactions, forming a molecule asymmetric in which different domains are located structural.

The cells have different receptors of specific membranes that recognize peptide sequences and / or functional domains of the laminin molecule. These receivers They can be classified into two groups: integrins and non integrins.

The integrins are formed by two chains transmembrane polypeptides,? and?, in association not covalent These molecules are the receptors through which The cells adhere to the components of the extracellular matrix. Some of them also intervene in cell recognition cell. Each recognizes specific peptide sequences that they are present in the matrix molecules, such as the laminin

Among non-integrin type receptors, the most studied is the receptor for 67 kDa for laminin, and it has been isolated and identified from several cellular tissues, between which are the carcinomas. In addition, it has been verified that metastatic tumor cells express on their cell surface more laminin receptors than normal cells, which is why, this receptor could be considered a marker of progression tumor and an indicator of the aggressiveness of many types of tumor.

Laminin presents several activities metastatic, such as:

- produce cell adhesion, growth and the extension;

- Stimulate the distinction between cells epithelial and tumor;

- cause cell migration;

- facilitate the malignancy of the cells tumor by its presence on the surface, which increases its invasive and metastatic activity.

Certain studies have shown that different functions of laminin are mediated by sequences specific peptides present in the laminin molecule, such as: the five amino acid sequence SIKVAVS, which is found located in fragment PA22-2 of the α chain of the laminin. Specifically the most active area of this region It is the SIKVAVS pentapeptide.

Summary of the invention

At present, liposome studies are directed to their conduction towards or to the selection as target of tumor cells through the incorporation on the surface of ligand vesicles, such as antibodies, peptides and proteins, which can recognize and specifically bind with this type of cell (Allen TM, Austin, GA, Chonn, A., Lin, L., Lee, KC, Biochem. Biophys. Acta (1991), 1061, 56-63).

Thus, the objective of the present invention It consists of a new application for anticancer drugs encapsulated in liposomes, which present as characteristic main to be coated by lipopeptides from the structure of the laminins (especially the SIKVAVS sequence) in such a way that the liposomes thus prepared demonstrate a high selectivity with respect to tumor cells and therefore increases the effectiveness of the encapsulated anticancer drug.

Meaning of the abbreviations used in the invention

DXR:

Doxorubicin

PC:

Phosphatidylcholine

PL:

Hydrogenated Egg Phospholipids

PG:

Phosphatidyl Glycerol

CHOL:

Cholesterol

CHROM:

Chroman-6

       \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ + # \ hfil \ + # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 A \ + \ + Ala \ + \ + Alanina \ cr C \ + \ + Cys \ + \ + Cysteine \ cr D \ +
\ + Asp \ + \ + Aspartic acid \ cr E \ + \ + Glu \ + \ + Acid
glutamic \ cr F \ + \ + Phe \ + \ + Phenylalanine \ cr G \ + \ + Gly \ + \ +
Glycine \ cr H \ + \ + His \ + \ + Histidine \ cr I \ + \ + Ile \ + \ +
Isoleucine \ cr K \ + \ + Lys \ + \ + Lysine \ cr L \ + \ + Leu \ + \ +
Leucine \ cr M \ + \ + Met \ + \ + Methionine \ cr N \ + \ + Asn \ + \ +
Asparagine \ cr P \ + \ + Pro \ + \ + Prolina \ cr Q \ + \ + Gln \ + \ +
Glutamine \ cr R \ + \ + Arg \ + \ + Arginine \ cr S \ + \ + Ser \ + \ +
Serina \ cr T \ + \ + Thr \ + \ + Threonine \ cr V \ + \ + Val \ + \ +
Valine \ cr W \ + \ + Trp \ + \ + Tryptophan \ cr Y \ + \ + Tyr \ + \ +
Tyrosine \ cr}
    

Lam2M:

myristoyl-PEGAD

Lam9:

Myristoyl-YESIKVAVS

Lam9Cys:

myristoyl-CYESIKVAVS

Lam9Cys-b-Ala:

myristoyl-AAAAACYESIKVAVS

AG10:

GYSRARKEAASIKVAVSARKE

(E8) -2-4G:

NPWHSIYITRFG

mir:

myristoyl

DOX:

doxorubicin

Detailed description of the invention

The present invention relates to the Preparation and use of liposomes containing drugs anti-cancer

The liposomes of the present invention have as its main feature that are coated with hydrophobically derivatized peptide fragments (lipopeptides coated) in such a way that the liposome so prepared offers a high selection capacity as a target with respect to tumor cells, thus increasing the effectiveness of the drug encapsulated anticancer.

Surprisingly, it has been shown that active in vitro coated lipopeptides before being incorporated into liposomes, would completely lose their ability to recognize when incorporated into liposomes, which is why, according to the invention, peptide spacers have been developed which, intercalated between the sequence The lipophilic chain of the coated lipopeptide would rarely maintain the ability to select as a target of the active peptide sequence.

In this way, the structure of the lipopeptide coated (hydrophobically derived peptide) is as follows:

Fragment lipid spacer sequence active

Therefore, the purpose of this invention relates to the preparation and use of liposomes that contain anti-cancer drugs that have on their surface Laminin derivatized peptide fragments (lipopeptides coated), made of the following three structural blocks: a lipid fragment, an active oligopeptide and a spacer oligopeptide among other fragments.

Lipid fragments are fatty acids of carbon chain length between C6 and C20. More specifically decanoyl, myristoyl and stearoyl.

The spacer fragment consists of inactive oligopeptides with respect to laminin having a length that lies between five and ten residues of amino acids. More specifically, with a length of seven to nine amino acid residues, and more specifically the sequences AAAAACYE, SSAAACYE and RKERKECYE.

The active sequence consists of the oligopeptide. SIKVAVS.

Liposomes that form liposomes are known well. Phospholipids are generally included, with net neutral charge or negative, and a sterol, like cholesterol. The choice of Lipids are performed based on the requirements regarding the size of the final liposome, the drug to be encapsulated and the Desired stability for preparation. Normally the component The largest lipid of the liposomes is phosphatidylcholine (PC). PCs distinguish each other in the length and degree of saturation of its acyclic chains and can be isolated from sources natural or synthesized The inclusion of a charged phospholipid negatively favors the stability of the liposome solution and prevents spontaneous aggregation of liposomes.

The negatively charged phospholipids more employees are phosphatidylglycerol (PG), phosphatidylserine (PS) phosphatidylinositol (PI), among others. The proportion used, of neutral phospholipid with respect to the negatively charged phospholipid It varies from 10: 2 to 10:10, respectively. The inclusion of a cholesterol generally favors liposome stability by cause decreased membrane permeability with with respect to ions and small polar molecules and it is also reduced the penetration capacity of a series of proteins between bilayers that could result in greater disorder between these. Normally, the proportion of cholesterol used amounts to from 0 to 50% of total lipids.

Optionally, the liposomes subject to This invention may contain additives that allow improving its stability properties or reduce drug toxicity encapsulated For example, mention may be made of the inhibitors of lipid oxidation, such as those described in US Pat. 5605703, EP 0274174, WO-8500968 and WO 9202208.

Anticancer drugs that can encapsulated in the liposomes of the present invention include, but they are not limited to:

Nitrogenous mustard analogs such as cyclophosphamide; melfalano; ifosfamide; or trophosphamide;

Ethylenimines as a thiotepa;

Nitrosoureas as carmustine;

Agents alkylating agents such as temozolomide; or dacarbazine;

Antimetabolites folic acid analogs such as methotrexate or raltitrexed;

Analogues of purines such as thioguanine, cladribine or fludarabine;

Analogues of pyrimidines such as fluorouracil, tegafur or gemcitabine;

Alkaloids of the vinca and analogues such as vinblastine, vincristine or vinorelbine;

Derivatives podophyllotoxin such as etoposide, taxanes, docetaxel or paclitaxel;

Anthracyclines and similar as doxorubicin, epirubicin, idarubicin and mitoxantrone;

Others cytotoxic antibiotics such as bleomycin and mitomycin;

Composed of platinum such as cisplatin, carboplatin and oxaliplatin;

Antibodies monoclonal like rituximab;

Other antineoplastic agents such as pentostatin, miltefosina, estramustina, topotecano, irinotecano and bicalutamide

As described above, the Liposomes of the present invention have the following features:

a) A lipid concentration of 1 and 100 mg / ml, and preferably about 10 mg / ml.

b) The lipid components are phospholipids, of both synthetic and natural origin, and cholesterol.

c) The proportion of cholesterol, with respect to the amount of total lipids is between 0 and 50%, preferably between 35 and 50%.

d) The phospholipids present are phosphatidylcholine, which has no net charge, and optionally another, negatively charged phospholipid, preferably phosphatidyl glycerol.

e) The ratio of the neutral phospholipid with respect to the negatively charged is between 10: 2 and 10:10 and preferably between 10: 7 and 10:10 respectively.

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f) Optionally liposomes may contain other additives such as oxidation inhibitors of lipids such as those described in US patents 5605703, EP 0274174, WO-8500968 and WO-9202208.

g) The concentration of peptide would vary between 0.1 and 1 mg / ml, and preferably about 0.5 mg / ml.

h) Liposomes are formed in a solution aqueous, buffered or not, physiologically isotonic. For example, 0.9% NaCl.

i) The size of the liposomes should be in any case less than 500 nm, and preferably less than 300 nm and more specifically between 50 nm and 250 nm.

Liposome preparation and drug incorporation

A preferred method is that presented by Bangham et al . in which heterogeneous multilamellar liposomes (MLV) are obtained in size. In this method, the lipids that are formed are dissolved in a suitable organic solvent that is subsequently removed by rotary evaporation under vacuum. The formed lipid film is subjected to hydration with a suitable aqueous medium containing the drug, by means of manual or mechanical agitation. The heterogeneous MLV suspension is subjected to any of the known procedures for size reduction and homogenization. For example, two preferred procedures are sonication with titanium probe to obtain SUV liposomes and extrusion through polycarbonate filters of the MLV solution to obtain VET liposomes.

Preparation of the peptide fragments

The synthesis of the peptides is carried out using the solid phase method of Merrifield (1962) with strategy Fmoc / tBu.

Lipopeptide Incorporation

The lipopeptides used were acyl oligopeptides, preferably the group acyl with linear saturated hydrocarbon chains of length C6 to C20 (preferably decanoyl, myristoyl or stearoyl). The lipopeptides were mixed with the rest of the components that were to constitute the liposomes, or were incorporated into the liposomes by incubation at 60 ° C of these lipopeptides and the vesicles, since, as the bilayers were in a gel state, allowed the incorporation of the hydrophobic part of these derivatives inside. In both cases, the hydrophobic zone of the derivatives it should remain forming the bilayer while the sequence Peptide would be hydrophilic on the outside.

By way of illustration, but not restrictively, the procedure described below in the present document detailed in the present patent by several examples practical.

Example 1 Synthesis of active peptides with carboxylic end

The synthesis of laminin-derived peptides is carried out following the solid phase method of Merrifield (1962) with Fmoc / tBu strategy.

To get an end sequence carboxylic, as the solid synthesis support, a Wang resin with a functionality grade of 0.72 meq / g resin who underwent the treatment described in the table below:

TABLE 1 Washing protocol for peptidyl resin

Stage Reagent Repetitions Weather one DMF 3 times one minute 2 Dichloromethane 3 times one minute 3 Amyl alcohol tertiary 3 times 1 minute 4 Ether until dried -

In general, the starting point was 1 gram of Wang resin in a syringe with a filter attached to a system vacuum, and was dimethylformamide (DMF) blown for 30 minutes. In parallel, weighed on a filter scale, the amount needed of the first Fmoc-amino acid and dissolved in DMF, adding coupling agents to this solution 4-dimethylaminopyridine (4-DMAP) and diisopropylcarbodiimide (DIPCDI) (0.3: 1, molar). All were used reagents in excess of 5 times with respect to the quantity required to complete the reaction. Then, this mixture was added to the previously drained resin and allowed to react for 2 hours at room temperature with occasional agitation. After this time the resin was washed with different solvents until drying full.

Finally, the total amount of amino acid In cases where the reaction was incomplete, it they added more reagents, in an amount corresponding to half of the initial amount used, and allowed to react for two more hours, repeating the same procedure thereafter resin drying and quantification of the incorporated amino acid.

The binding of Fmoc-amino acids remaining was carried out through successive stages of deprotection of the amino group and formation of the amide bond.

Thus, for the suppression of the Fmoc protective group amino, the peptidyl resin was treated once with DMF / 20% piperidine for one minute, repeating the treatment A second time for 5 minutes. Then, piperidine was removed with several DMF washes and the test of ninhydrin to verify complete elimination of the Fmoc group (blue coloring) In some cases, deprotection was performed with the reagent 1,8-diazabicyclo [5.4.0.] - undec7-eno (DBU) used in the DMF / piperidine / DBU mixture (48: 2: 2, v / v / v) by medium of a single resin treatment for 7 minutes. In the end from this time, the resin was washed several times with DMF and performed the ninhydrin test again precisely as in the case previous.

Once he checked out the peptidyl resin, the Fmoc-relevant amino acid and reagents of coupling Depending on the difficulty of the sequence synthesized presented, two different combinations of reagents:

HOBt and DIPCDI, in the 1: 1 molar ratio, with the Fmoc-amino acid.

HOBt, DIEA and tetrafluoroborate 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium (TBTU), in the 1: 2: 1 molar ratio.

All reagents were used in excess of 2.5 times with respect to the necessary quantity.

In both cases the reaction was left during a hour, controlling the conclusion by means of the ninhydrin test for the disappearance of free amino groups (coloration yellow). If the reaction was not complete, the mixture was left in resin contact for one more hour, after which it I repeated the ninhydrin test. In case there was still free amino groups in the resin, it was washed several times with DMF and reagents were added again in half the amount initially used On some occasions, and still repeating the reaction, incomplete couplings occurred. To continue with the synthesis without anomalous chains forming, it was it is necessary to block incomplete chains by acetylation of the amino groups that were still free. To this end, the resin with 2 mequivalents of acetic anhydride and 1 mequivalent of 4-DMAP for each mequivalent of peptidyl resin for 30 minutes. Then washed with DMF and a ninhydrin test was performed to verify the total disappearance of amino groups (yellowing).

The ninhydrin test was replaced by Chloranil test in the case of detecting secondary amino groups of amino acids such as proline, since ninhydrin does not react with these groups.

Example 2 Synthesis of peptides with amino terminal end

Carboxamide end peptides are obtained from the resin p-methylbenzhydrylamine (MBHA). This resin needs a special initial treatment that includes several washes with a 40% DCM / TFA acid mixture, leaving finally in contact with the resin for 20 minutes. After, To remove the acid, it was washed 5 times with DCM for 1 minute each time, and to neutralize it, the resin was treated with the mixture 5% DCM / diisopropylethylamine (DIEA) base, until found that the pH of the resin was basic. Finally, to eliminate the DIEA, washed several times with DCM.

Then, the coupling of the acid spacer p - [(R, S) -alpha [1-9H-fluorene-9-e) -methoxyformamide] -2,4-dimethoxybenzyl] -phenoxyacetic (AM), protected with the Fmoc group, which is what it provides to the sequence your extreme amide. For this, the Fmoc-AM in excess of 1.5 times the amount required, and added to the resin together with the reagents hydroxybenzotriazole (HOBt) and DIPCDI (1: 1, molar), also in excess, letting the reaction take place for 90 minutes. Be determined the conclusion of the reaction by means of the test of Kaiser or ninhydrin test, verifying the disappearance of groups resin free amino. In the event that all spacers did not join, the reaction would be repeated once more, using the half of the initial amount of reagents used. Once all spacers joined together, the resin was washed several times with DMF to remove excess reagents.

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The coupling of Fmoc-amino acids remaining was performed through successive stages of deprotection of the amino group and formation of amide bond, precisely as described in example 1.

Example 3 Deprotection and de-anchoring of the peptide

For deprotection of the peptide sequence free, the Fmoc group from the amino end is removed first terminal following the protocol given in the following table:

TABLE 2 Deprotection and de-anchoring protocol of peptide

Stage Reagent Repetitions Weather one DMF 3 times one minute 2 DMF / piperidine al twenty% 1 time 1 minute 3 DMF / 20% piperidine 3 times 5 minutes 4 DMF 3 times 1 minute 5 DCM 3 times 1 minute 6 Tertiary amyl alcohol 3 times 1 minute 7 Ether Until dried

In some syntheses the DMF / 20% piperidine with the DMF / piperidine / DBU mixture (48: 2: 2, v / v / v), which is left in contact with the peptidyl resin for 7 minutes.

Then the peptide was removed from the resin and removed the protective groups from the functional chains of amino acid, in a single stage. To achieve this, they prepared several mixtures of TFA with different scavengers as anisole, thioanisole, phenol, mercaptoethanol and water, according to the groups protectors present in peptide chains. It weighed a aliquot of the peptidyl resin in a syringe with a filter coupled to a vacuum system and the mixture was added acid from eliminators, leaving in contact with the resin for 2 to 3 hours, at room temperature with additional agitation. A After this time, the resin was filtered and washed 3 times with TFA, collecting the filtrates and the products washed in a tube. First, the TFA was removed by evaporation with nitrogen and then cold diethyl ether was added, obtaining a white precipitate (free peptide). The precipitate was centrifuged at 3000 rpm for 15 minutes, removing the supernatant by drained and repeated the procedure 5 more times. Finally I know they removed the traces of ether from the solid with nitrogen, it was returned to dissolve in water or 10% acetic, depending on the solubility of the peptide, and lyophilized to obtain the free peptide product completely dry gross.

Example 4 Hydrophobic derivatization of phase peptide sequences solid

Fatty acids were coupled to the sequences in the same way as Fmoc-amino acids, by means of forming an amide bond with the carboxylic group of fatty acid.

Thus, an aliquot of the peptidyl resin in a syringe with filter connected to a vacuum pump and swelled with DMF. Then the Fmoc group deprotection. Once unprotected, the fatty acid used in each case, in excess of 2.5 times, together with DPCDI / HOBt or, TBTU / DIEA / HOBt synthesis reagents, depending on the peptide sequence in question. The conclusion of the reaction, precisely during the synthesis, by means of the ninhydrin test for the disappearance of groups amino free.

To obtain the free hydrophobic derivative, treated the peptidyl resin with the same acid mixture of TFA and eliminators, and under conditions identical to those used in the de-anchoring of the initial peptide sequence.

Example 5 Obtaining liposomes containing doxorubicin and a lipopeptide coating the surface of the liposome

Initially, and in all cases, it they prepared large multilamellar liposomes (MLV) following the method described by Bangham. From this, and by sonication, small unilamellar liposomes were obtained (SUV)

All the material and solutions used were sterile and, throughout the procedure, the work was carried out in a laminar flow hood to maintain the sterility.

Liposomes prepared with derivatives Hydrophobic of the two active sequences had in their composition: phosphatidylcholine (PC), phosphatidylglycerol (PG), cholesterol and chromano-6. The following procedure was adopted to get them:

Thus, first, liposomes were prepared SUV PC, PG, cholesterol and cholesterol were weighed chromano-6, and dissolved in chloroform, the solvent being removed by evaporation in the rotary evaporator in order to form a lipid film. Any deleted trace of solvent that could be left by lyophilization that lasts 1 hour

After this period, the hydration was hydrated. film with 1 mL of 0.9% NaCl, keeping the container spherical in a bath at 60 degrees Celsius for 1 hour. They were added 1.2 mL of a solution of doxorubicin to MLV liposomes obtained, having a concentration equal to 2 mg / mL (2.4 mg). Be let the preparation stand for 15 minutes in a 60 bath degrees Celsius and then the spherical vessel was stored in a vacuum-free rotary evaporator that turned slowly during a 20 minute period.

In order to obtain SUV liposomes, it they subjected the MLV to sonication in an ultrasonic bath for 8 cycles lasting each 2 minutes, separated by intervals of 5 minutes of rest in a bath at 60 degrees Celsius.

The incorporation of lipopeptides led to out by mixing an aliquot of 200 µL of liposomes, 200 µL 0.9% NaCl and 12 µL of a solution of lipopeptide in DMSO (c = 10 mg / mL). The mixture was allowed to stand at 60 degrees Celsius for an hour and then at room temperature for 30 minutes plus.

Alternatively, liposomes were prepared incorporating the lipopeptide from the beginning. So, they mixed PC, PG, cholesterol and chromano-6 lipids with a aliquot of the lipopeptide dissolved in chloroform / methanol, in it molar ratio than in the previous case. The rest of the procedure is identical to the previous case.

Finally, to eliminate doxorubicin no encapsulated and the unincorporated lipopeptide, the sample was placed in a PD-10 column (Sephadex G-25). For this, the column was first equilibrated with 0.9% NaCl. Once equilibrated, the sample was added, which was also eluted with 0.9% NaCl, until it overflowed the column. The volume of Liposomes obtained were completed up to 2 mL.

After this procedure, the following types of liposomes incorporating doxorubicin:

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Composition Conc. Of Conc. from Lipopeptide Coating Conc. from Size of the lipid lipids drug peptide liposome PC / PG / col. / Cro 9.91 mg / mL 1.04 mg / mL Myristic- (A) 5 -CYESIKVAVS 0.42 mg / mL 160 nm PC / PG / col. / Cro 14.05 mg / mL 1.5 mg / mL Myristic-PEAGD 1.1 mg / mL 115 nm

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Example 6 Obtaining liposomes containing paclitaxel and a lipopeptide lining the surface of the liposome

Initially, and in all cases, it they prepared large multilamellar liposomes (MLV) following the method described by Bangham. From them, and by sonication, small unilamellar liposomes were obtained.

All the material and solutions used were sterile and, throughout the procedure, the work was carried conducted in a laminar flow campaign to maintain the sterility.

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Liposomes prepared with derivatives Hydrophobic active sequences had in their composition: phosphatidylcholine (PC), phosphatidylglycerol (PG) and cholesterol. Be adopted the following procedure to obtain them:

Thus, first, liposomes were prepared SUV The PC and cholesterol were weighed, and dissolved in chloroform, removing the solvent by evaporation in the rotary evaporator to form a lipid film. It was deleted any trace of solvent that could be left by lyophilization that lasts 1 hour.

After this period, the hydration was hydrated. film with 1 mL of 0.9% NaCl, keeping the container spherical in a bath at 60 degrees Celsius for 1 hour. They were added 1.2 mL of a solution of paclitaxel to the MLV liposomes obtained, having a concentration equal to 0.5 mg / mL (0.6 mg). It was left in stand the preparation for 15 minutes in a 60 degree bath Celsius and then the spherical vessel was stored in an evaporator vacuum-free rotary rotating slowly during a period of 20 minutes.

To obtain SUV liposomes, the MLV to sonication in an ultrasonic bath for 8 cycles lasting each 2 minutes, separated by 5 minute rest intervals in a bath at 60 degrees Celsius.

The incorporation of lipopeptides led to out by mixing an aliquot of 200 µL of liposomes, 200 µL 0.9% NaCl and 12 µL of a solution of lipopeptide in DMSO (c = 10 mg / mL). The mixture was allowed to stand at 60 degrees Celsius for an hour and then at room temperature for 30 minutes plus.

Alternatively, liposomes were prepared incorporating the lipopeptide from the beginning. So, they mixed PC, PG and cholesterol lipids with an aliquot of the lipopeptide dissolved in chloroform / methanol, in the same molar ratio as in the last case. The rest of the procedure is identical to the case. previous.

Finally, to eliminate paclitaxel no encapsulated and the unincorporated lipopeptide, the sample was placed in a PD-10 column (Sephadex G-25). For this, the column was first equilibrated with 0.9% NaCl. Once equilibrated, the sample was added, which was also eluted with 0.9% NaCl, until it overflowed the column. The volume of Liposomes obtained were completed up to 2 mL.

After this procedure, the following types of liposomes incorporating paclitaxel:

Composition Conc. Of Conc. from Lipopeptide Coating Conc. from Size of the lipid lipids drug peptide liposome PC / PG / col. 8.93 mg / mL 0.26 mg / mL Myristic- (A) 5 -CYESIKVAVS 0.42 mg / mL 140 nm PC / PG / col. 13.5 mg / mL 0.4 mg / mL Myristic-PEAGD 1.1 mg / mL 105 nm

Example 7 Cell adhesion tests

Solutions of laminin-1 and synthetic peptides (50 mg / well) in 96-well tissue culture plate wells of TPP (Switzerland). The wells were dried at room temperature overnight. Before use, the wells were washed with saline. Buffered free of calcium and magnesium ions. The locks were blocked. remaining free radicals of polystyrene using a solution of 1% BSA.

They were cultured and labeled with 51 Cr cells of human fibrosarcoma HT1080. Marked cells were placed (1 cpm / well) in wells containing laminin and peptides synthetic

After 30 minutes of incubation at 37 degrees Celsius, the non-adherent cells were removed by washing. Be scraped the adhered cells and the radioactivity was measured. Be show the specific percentages of adhesion found in the Figure 1 attached.

Example 8 Inhibition of cell adhesion to laminin (complete molecule) in vitro by laminin peptides

Following the example described in the procedure of example 1 cells 1 adhered H5-1080 labeled with 51 Cr, in wells (0.32 cm2) coated with 1 µm of laminin. The were incubated adhered cells with different concentrations of fragments synthetic laminin peptides. The results obtained are shown in the attached figure 2.

Example 9 Antiproliferative effect of targeted doxorubicin liposomes against specific receptors for laminin peptides in cells tumor

The antiproliferative effect of doxorubicin following the MTT method. The cells were seeded HT1080 obtained from exponential cultures in 0.36 wells cm2 (96-well tissue culture plates of TPP, Switzerland) with a density of 5000 cells per well. One day later, it they washed the cells and incubated for two hours with liposomes containing doxorubicin The formations were adapted different liposomes at the same drug concentration and the test was carried out in parallel wells (increasing the doxorubicin concentration from 0.01 µg / ml to 10 \ mug / ml). After incubation, the cells were washed five times with PBS and incubated for three days in a complete medium. After this period, 50 µL of PBS was added to each well which contained 1 mg / ml MTT (tetrazolium salt, Sigma) and incubated for four additional hours. The DMSOs were dissolved in intracellular crystals of formazan resulting from the reduction of tetrazolium salt, only present in active cells. Be calculated the number of metabolically active cells by measuring the absorbance of this DMSO solution at 540 nm.

Activity percentage was calculated cytostatic according to formula (A-B) / Ax100, in which A is the absorbance in tumor cells incubated in a medium of control and B is the absorbance in tumor cells incubated with the Liposome preparations

The results of cytostasis are shown resulting in the attached figure 3, in which:

- the results represent the mean +/- the standard deviation of the three independent experiments performed in triplicate;

- IC50 is defined as the concentration of drug in which 50% of cells survive in comparison with the control lot; Y

- P> 0.05; Student's t test.

Example 10 Biodistribution of doxorubicin administered as a free drug or preparation of liposomes (PC / PG / Col / miris- toílo-AAAAACYESIKVAVS) / doxorubicin) in animals with tumors

Animals : Tests were performed on nude and immunosuppressed BALB / c mice obtained from the animal production area of IFFA CREDO Inc. (Lion, France). Animals were stored in laminar flow cabinets under pathogen-free conditions and used when they reached an age of 8 weeks.

Cell culture conditions : Human fibrosarcoma HT1080 cells were grown in Ham's F-12 medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine , and vitamin solution (GIBCO, Grand Island, NY). The cultures were stored in plastic and incubated in 5% CO2 - 95% air at 37 degrees Celsius in humidified incubators. The cell line was examined to certify the absence of mycoplasma.

Tumor cells were collected from subconfluent cultures (50-70% confluence) by treatment with trypsin (0.25%) and EDTA (0.02%). The cells were washed in a supplemented medium and then resuspended in Hank's balanced salt solution (HBSS) for subsequent injection. Only monocellular suspensions with a viability of more than 90% (determined by trypan blue staining) were used for in vivo studies.

Biodistribution test : HT-1080 cells were premixed at a concentration of 1x10 7 cells / mL of HBSS with an equal volume of Matrigel liquid (Collaborative Biomedical Products, Bedford, MA) at 10 mg / mL. 0.02 mL of the resulting suspension was inoculated subcutaneously in the left slit of the mouse. Tumor growth was monitored twice a week. When the tumors reached a volume of 1 cm 3 (day 25 after injection of the cells), the mice received a single intravenous dose of doxorubicin (5 mg / kg) as a liposome preparation or free drug. At times of 30 minutes, 5 hours and 24 hours after drug administration, the mice were sacrificed and samples of tumor tissue and plasma were taken. The results obtained in the attached figures 4 and 5 are shown.

Claims (17)

1. Liposomes that encapsulate anticancer drugs, characterized in that they are coated with a lipopeptide composed of three substructures: a lipid fragment, an active oligopeptide and an oligopeptide spacer between the other two fragments. 2. Liposomes encapsulating anticancer drugs according to claim 1, characterized in that the lipid fragment of the coating lipopeptide is fatty acids of carbon chain length between C6 and C20. 3. Liposomes encapsulating anticancer drugs according to claims 1 and 2, characterized in that the lipid fragment of the coating lipopeptide is preferably decanoyl, myristoyl or stearoyl. 4. Liposomes encapsulating anticancer drugs according to claims 1 to 3, characterized in that the active sequence fragment of the coating lipopeptide is SIKVAVS. 5. Liposomes encapsulating anticancer drugs according to claims 1 to 4, characterized in that the oligopeptide spacer between the active sequence fragment and the lipid fragment of the coating lipopeptide is an oligopeptide that is between five and ten amino acid residues in length. 6. Liposomes encapsulating anticancer drugs according to claims 1 to 5, characterized in that the oligopeptide spacer between the active sequence fragment and the lipid fragment of the coating lipopeptide is one of the following sequences: AAAAACYE, SSAAACYE or RKERKECYE. 7. Liposomes encapsulating anticancer drugs according to claims 1 to 6, characterized in that the ratio of the total lipids that form the liposomes with respect to the drug is between 20: 1 and 2: 1, and preferably is 10: 1. 8. Liposomes encapsulating anticancer drugs, according to claims 1 to 7, characterized in that the lipid components of the liposomes are phospholipids, both synthetic and natural, and cholesterol. 9. Liposomes encapsulating anticancer drugs according to claims 1 to 8, characterized in that the phospholipids present are preferably phosphatidylcholine, which has no net charge, and another negatively charged phospholipid, preferably phosphatidylglycerol. 10. Liposomes encapsulating anticancer drugs according to claims 1 to 9, characterized in that the ratio of the neutral phospholipid to the negatively charged is between 10: 2 and 10:10, preferably between 10: 7 and 10:10. 11. Liposomes encapsulating anticancer drugs according to claims 1 to 10, characterized in that the proportion of cholesterol, with respect to the total amount of lipids, is between 0 and 50% and preferably between 35 and 50%. 12. Liposomes encapsulating anticancer drugs according to claims 1 to 11, characterized in that they may optionally contain an additive for lipid peroxidation inhibition. 13. Liposomes encapsulating anticancer drugs according to claims 1 to 12, characterized in that the lipid peroxidation inhibition additives are vitamins and their derivatives such as vitamin E or vitamin E acetate, an antioxidant authorized for pharmaceutical use such as BHT or a chromane or chromene, such as 3,4-dihydride-2,2-dimethyl-6-hydroxy-7-methoxy-2H-1-benzopyran. 14. Liposomes encapsulating anticancer drugs according to claims 1 to 13, characterized in that the proportion of the coating lipopeptide with respect to the total amount of lipids is between 0.1% and 30%, preferably between 1% and the 15%. 15. Liposomes encapsulating anticancer drugs according to claims 1 to 14, characterized in that they have an average size that is between 50 nm and 250 nm. 16. Liposomes according to claims 1 to 15, characterized in that the encapsulated anticancer drugs are:

Analogues of the nitrogen mustard such as cyclophosphamide; melfalano; ifosfamide; or trophosphamide;

Ethylenimines as a thiotepa;

Nitrosoureas as carmustine;

Agents alkylating agents such as temozolomide; or dacarbazine;

Antimetabolites folic acid analogs such as methotrexate or raltitrexed;

Analogues of purines such as thioguanine, cladribine or fludarabine;

Analogues of pyrimidines such as fluorouracil, tegafur or gemcitabine;

Alkaloids of the vinca and analogues such as vinblastine, vincristine or vinorelbine;

Derivatives podophyllotoxin such as etoposide, taxanes, docetaxel or paclitaxel;

Anthracyclines and similar as doxorubicin, epirubicin, idarubicin and mitoxantrone;

Others cytotoxic antibiotics such as bleomycin and mitomycin;

Composed of platinum such as cisplatin, carboplatin and oxaliplatin;

Antibodies monoclonal like rituximab;

Other antineoplastic agents such as pentostatin, miltefosina, estramustina, topotecano, irinotecano and bicalutamide 17. Use of liposomes that encapsulate drugs anti-cancer, according to claims 1 to 16, for the preparation of a medicament for intravenous administration in humans or other mammals for the treatment of tumors malignant